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Posttranslational modification, folding, assembly, and death
1. Covalent modifications
1) Proteolytic processing: posttranslational processing is
called “maturation”. Presequences such as signal peptide
and other entities are
cleaved by proteases and
proteins thereby retain their
final structural information.
Examples… all secretory
proteins, proteins targeted
into chloroplast and some
other organelles, signaling
proteins such as prosystemin,
insulin, etc.
2) Protein phosphorylation
Arguably the most important posttranslational modification of
proteins in eukaryotic cells.
Which amino acid residues are phosphorylated?
Protein kinases and phosphatases. Their names and substrate
specificity. Plants vs. animals—differences will be further covered in
signaling lectures
2) Acetylation: N-terminal amino acid attached by an acetyl group
3) Fatty acid or hydrocarbon
chain addition---talked about
in membrane lecture
(myristoylation,
prenylation)…
4) Disulfide bond changes catalyzed by PDI
5) Glycosylation—sugar addition.
What residues to be glycosylated? Asp and Asn—O-linked or N-linked
reactions happen in different places (Golgi and ER)
2. Protein folding
The AA sequence of protein - 3_D structure
1) Spontaneous or assisted process? Evidence?
2) Molecular chaperones : proteins that bind and stabilize
other proteins and help them fold/assemble properly (can be
folding of one protein and assembly of multiple proteins).
Heat shock protein story:
Two major types: type I includes hsp70---bind and prevent misfolding of
the substrate proteins (can also unfold proteins)---cytosol,
chloroplast, mitochondria.
Type II also referred to chaperonins such as hsp60 (GroEL in bacteria)
family proteins that form large complex like a beer barrel that brew
the substrate proteins with the help of “lid” protein such as cpn10
in the chloroplast (or GroES in bacteria).
The crystal structure
of GroEL/S in
bacteria: central
cavity is where the
folding occurs. Double
rings each consists of
7 subunits (total of
14 subunits of
GroEL/hsp60). The lid
of the barrel is a
heptameric complex
of GroES/cpn10.
The first ring interacts
with the protein
substrate. ATP binding to
each of the ring subunits
triggers release of the
bottom lid that in turn
trap the substrate inside
the barrel and “brew” it
with more ATP. Binding of
ATP eventually trigger
release the substrates.
Every step involves
consumption of ATP---the
ring subunits are ATPases.
Cytoplasm made
proteins can be
folded by different
mechanisms
depending whether
it is soluble or on
the membrane (ER).
The cytosol
proteins can go
through both type
1 and type 2
chaperones but ER
proteins are
typically folded by
type 1.
3) Protein foldases---enzymes that catalyze folding
PDI—protein disulfide isomerase catalyze correct formation
of S-S_bonds as described earlier.
PPIase: peptidyl prolyl isomerase involved in rotating the peptide
bond between any amino acid and proline—one of the rate limiting
steps in folding process.
3. Protein assembly
Many proteins function in multisubunit complex. Assembly from
single subunits---functional complex. Examples: Rubisco—how
chloroplast chaperones were found---Rubisco LSU-binding
proteins.
How one protein
recognize the
others for
assembly?
Specific vs nonspecific
interaction:
Domains and
modules: “lock and
key”
4. Protein degradation—end of the life cycle
1) Biologically very important: Examples, a regulatory protein
works when it is needed. Degradation is a way to remove the
“function”. Such function can be positive or negative…
2) Where degradation occurs: many compartments inside the cell.
3) What enzymes:a large number of proteases, some cleave a specific
bond some chew the entire protein.
4) How it is done: VERY tightly regulated and specific degradation!
Need to pick out one from 10,000 in the cell…
Ubiquitin-tagging of proteins: serves as signal for degradation by
proteasome, a large
multisubunit complex--degradation machine with
similar structure to
chaperonin machine!
Ubiquitin is a small protein
That is covalently attached
to the lysine residue of a
Protein via its C-terminus.
Poly-ubiquitin chain can be
Formed by the same link
Between ubiquitins.
The ubiquitin-conjugation
involves three enzymes
called E1 E2 E3. E1 and
E2 “activate” Ubi protein
and E3 interacts with both
substrate and Ubi to
transfer the Ubi to the
substrate. Repeat Ubi
addition step to form the
Ubi chain. At least 4 Ubi
units are required for
delivery to proteasome.
The E2/3 enzymes will be
released after the Ubi
chain formation. The Ubi
chain will be recognized by
the proteasome and the
substrate is sent for
degradation but Ubi is
“spit” out of the
proteasome for reuse.